The transcription factor NF-kB extensively regulates a plethora of genes and is involved in cancer and inflammatory disease. Aberrant constitutive NF-kB activation has been recognized as a critical pathogenetic in cancer and inflammatory disease. Our long-term goal is to elucidate the mechanism controlling specific expression of NF-kB target genes. The specific hypothesis is that NF-kB DNA binding complexes contain novel components, besides the Rel dimer and these mediate specific gene regulation. We based that hypothesis on the observations 1) the original size estimate of native NF-kB in nuclear extracts was > 200 kD, but when reconstituted from purified p50 and p65 proteins, it was estimated to be 115 kD which would be appropriate for a simple heterodimer, 2) reconstituted heterodimers from purified proteins have a > 100 fold lower affinity than native NF-kB, 3) our recent finding that ribosomal protein S3 (RPS3) is a novel component in NF-kB DNA binding complexes that mediates selective gene regulation. The experimental focus of this proposal is to identify the novel subupjtsgfNFTkB andsto define the biochemical function of rion-Rgl ^ suburiits. The specific aims are: 1. to elucidate the mechanism of RPS3-mediated specificity in NF-kB target gene regulation; 2. to identify novel component(s) in NF-kB DNA binding complexes that mediated specific gene expression. We will purify the novel components in CD25 kB DNA binding complex, elucidate the functional significance and mechanism of novel components in NF-kB signaling under normal and pathological settings; 3, to develop in vivo models and design novel small peptides targeting RPS3 and novel components in NF-kB DNA binding complexes to specifically irihibitor NF-kB genes. We will map the minimal parts in p65, RPS3 and novel components for physical interaction and NF-kB function and assess the inhibitory capability of cell-permeable peptides targeting at these minimal parts. This project will provide new insight into NF-kB and better our understanding of the regulatory specificity of NF-kB. These results will also provide potential novel therapeutic targets for the treatment of cancer and inflammatory disease, and may have implication for NF-kB-related diseases in general.